Thin film cell encapsulation devices

ABSTRACT

Thin film devices, e.g., multilayer thin film devices, that encapsulate cells for transplantation into a subject are provided. Also provided are methods of using and methods of preparing the subject devices. The thin film devices include a first porous polymer layer and a second porous polymer layer that define a lumen therebetween and encapsulate a population of cells within the lumen. The thin film devices can promote vascularization into the lumen of the device via the pores in the first polymer layer and/or second polymer layer; limit foreign body response to the device; limit ingress of cells, immunoglobulins, and cytokines into the lumen via the first and the second polymer layers; and release from the first polymer layer and/or the second polymer layer molecules secreted by the population of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/111,773, filed Aug. 24, 2018, which is a continuation ofU.S. patent application Ser. No. 15/713,098, filed Sep. 22, 2017, issuedas U.S. Pat. No. 10,087,413, which is a continuation ofPCT/US2016/023808, filed Mar. 23, 2016 which claims the benefit of U.S.Provisional Patent Application No. 62/136,997, filed Mar. 23, 2015, thedisclosures of which are herein incorporated by reference.

INTRODUCTION

Cell replacement therapy has seen unprecedented progress in the past fewyears, including the ability to achieve insulin independence in humansthrough islet transplantation (Atkinson et al., Lancet 2014, 383, 69-82;Orlando et al., Diabetes 2014, 63, 1433-1444; Hatziavramidis et al.,Ann. Biomed. Eng. 2013, 41, 469-476). Advancements in stem celltechnology hold potential to overcome donor shortages for many patients,who can benefit from islet replacement therapy. In particular,stem-cell-derived beta cells offer a promising new cell source forachieving insulin independence. Unfortunately, life-long systemicimmunosuppression is required to protect transplanted cells from beingrejected putting patients at risk of organ damage, infection andmalignancies (Ludwig et al., Proc. Natl. Acad. Sci. U.S.A 2013, 110,19054-19058; Shapiro et al., N. Engl. J. Med. 2000, 343, 230-238). Cellencapsulation provides an alternative approach to protect transplantedcells without the complications associated with immunosuppression. Whilea number of strategies are being investigated (Huang et al., PLoS One2013, 8; Shah, Biomatter 2013, 3, 1-7; Song et al., Mater. Sci. Eng. C.Mater. Biol. Appl. 2014, 40, 197-203; De Faveri et al., Neuroeng. 2014,7, 7; Robles et al., Cell Transplant. 2013; Tomei et al., Proc. Natl.Acad. Sci. U.S.A 2014, 111, 10514-10519), there are several challengesassociated with these approaches: retrievability, control over poredimensions, biocompatibility, scalability, and reproducible fabricationmethods.

The key function of an encapsulation device is to create an environmentthat allows for normal insulin secretion in response to fluctuatingblood glucose, while maintaining cell viability through sequestrationfrom the immune system and effective nutrient and waste exchange. Withthe goal of creating immune-protected beta cells, a variety of micro-and macro-encapsulating approaches have been developed over the pastseveral decades (Scharp et al., Adv. Drug Deliv. Rev. 2014, 67-68,35-73; Weir, Diabetologia 2013, 56, 1458-1461; Buder et al., ImmuneNetw. 2013, 13, 235-239, Julien et al., Fontiers Biosci. (Landmark Ed.)2014, 19, 49-76). The fundamental distinction between micro- andmacro-devices is a matter of scale: micro-encapsulation approachesencapsulate a single cell or islet, which maximizes surface area tovolume ratios and promotes improved nutrient exchange (Cala et al.,Clinical Application of Microencapsulated Islets: Actual Prospectives onProgress and Challenges. 2014, 68, 84-92; Cornolti et al., Cell 2009,18, 195-201). However, there is limited control of membrane thicknessand pore size with micro-encapsulation.

Additionally, since islets are individually encapsulated, thousands ofmicrodevices are required for each transplant, and capsule size makeslive imaging and tracking a significant challenge. Conversely,macro-encapsulation devices house many cells or islets (Lathuilière etal., Biomaterials 2014, 35, 779-791). These larger devices allow forgreater control over membrane parameters, such as pore size andporosity, but are plagued by limited nutrient diffusion and cellresponse due to the device thickness and large device reservoir. Inaddition to these challenges, the sharp rigid structures typicallyassociated with macro-encapsulation devices can lead to a foreign bodyresponse and subsequent device failure from fibrotic encapsulation(Ward, J. Diabetes Sci. Technol. 2008, 2, 768-777; Ward et al.,Biomaterials 2002, 23, 4185-4192).

The present disclosure address these as well as other needs in the fieldof delivery of biological material to a subject in need thereof.

SUMMARY

Thin film devices that encapsulate cells for transplantation of thecells into a subject are provided. Also provided are methods ofpreparing the subject devices and methods for using the subject devices.The thin film devices include a first layer, a population of cells and asecond layer, where the first layer and the second layer are in contactwith one another around the periphery of the cells disposed between thefirst layer and the second layer. The thin film medical devices are usedin methods for providing encapsulated transplanted cells in a subject.The method includes transplanting the thin film device into a subject;promoting vascularization of the device and/or; inhibiting foreign bodyresponse and/or; limiting ingress of cytokines, immunoglobulins andcells into the device; and releasing molecules secreted by theencapsulated cells.

In certain embodiments, a method for transplanting cells in a subject isdisclosed. The method may include: (a) providing a thin film devicecomprising: (i) a first nanoporous or microporous polymer layer; (ii) asecond nanoporous or microporous polymer layer, wherein the first andsecond layers define a lumen between the first and second layers; and(iii) a population of cells disposed in the lumen between the first andsecond layers, wherein first and second polymer layers are each lessthan 15 μm thick, and the device has a surface area of 1 cm² to 5 cm²,(b) transplanting the device into a subject, wherein one or more of: (i)vascularization into the lumen of the device via the pores in the firstpolymer layer and/or second polymer layer; (ii) a limited foreign bodyresponse to the device; (iii) a limited ingress of cells,immunoglobulins, and cytokines into the lumen via the first and thesecond polymer layers, is observed.

In certain embodiments, the method may further include releasing fromthe first polymer layer and/or the second polymer layer moleculessecreted by the population of cells.

In certain embodiments, the population of cells may survive for at leastone month after the transplanting.

In certain embodiments, the population of cells comprises pancreaticislet cells and wherein the pancreatic islet cells sealed inside thedevice respond to glucose level of the subject by secreting insulin. Incertain embodiments, the subject has or is predisposed to developingdiabetes.

In certain embodiments, the first and second polymer layers arenanoporous polymer layers and may each include a supporting layer, suchas, a microporous backing layer adhered to a surface of the nanoporouspolymer layer.

In certain embodiments, the first and second polymer layers aremicroporous polymer layers.

In certain embodiments, the first polymer layer is a nanoporous polymerlayer and the second polymer layer is a microporous polymer layer.

In certain embodiments, the first and second polymer layers arefabricated using the polymer poly-caprolactone (PCL).

In certain embodiments, the first and second polymer layers are sealedalong the entire periphery of the device thereby providing a closedlumen.

In certain embodiments, the lumen comprises an opening at which thefirst and second polymer layers are not sealed to each other.

In certain embodiments, the lumen comprises an opening at which thefirst and second layers are not in contact with each other, the devicefurther comprising a tubing comprising a first end and a second enddistal to the first end, wherein the first end of the tubing ispositioned in the opening and the second end is configured to introducethe population of cells into the lumen.

In certain embodiments, the lumen comprises a first opening and a secondopening at which the first and second layers are not in contact witheach other, the device further comprising a first tubing and a secondtubing, each of the first and second tubing comprising a first end and asecond end distal to the first end, wherein the first end of the firsttubing is positioned in the first opening and the first end of thesecond tubing is positioned in the second opening, and wherein thesecond end of each tubing is used for ingress or egress of fluids fromthe lumen.

In certain embodiments, the method comprises after the transplanting,attaching the second ends of the first and second tubing to a fill port.In certain embodiments, the fill port comprises a first chamber in fluidcommunication at the fill port with the second end of the first tubing;and a second chamber in fluid communication at the fill port with thesecond end of the second tubing.

In certain embodiments, a thin film device for transplanting cells in asubject is provided. The device includes a first nanoporous ormicroporous polymer layer; a second nanoporous or microporous polymerlayer, wherein the first and second polymer layers define a lumenbetween the first and second polymer layers; and a population of cellsdisposed in the lumen between the first and second polymer layers,wherein first and second layers are less than 15 μm thick, and thedevice has a surface area of 1 cm² to 5 cm², and wherein (a) the poresin the first and second polymer layers limit ingress of cells,immunoglobulins, and cytokines into the lumen via the first and thesecond layers, and/or (b) the pores are sized to promote vascularizationinto the lumen of the device, and/or (c) the device is configured tolimit foreign body response to the transplanted device, and (d) thepores are sized to release molecules secreted by the population ofcells. In certain embodiments, first and second polymer layers arenanoporous layers and may each include a supporting layer, such as, amicroporous backing layer adhered to a surface of the nanoporous polymerlayer.

In certain embodiments, a method for making a device disclosed herein isprovided. The method may include placing a first nanoporous ormicroporous polymer layer over a second nanoporous or microporouspolymer layer, wherein the first and second polymer layers are similarlydimensioned; sealing the first and the second polymer layers along theperiphery of the polymer layers leaving an unsealed area at a locationalong the periphery to provide an opening for access to a lumen definedbetween the first and second layers and the periphery of the polymerlayers at which the polymer layers are sealed together, placing apopulation of cells into the lumen through the opening; and sealing theopening. In certain embodiments, the first and second polymer layers aresealed together via application of heat. In certain embodiments, thefirst and second polymer layers are sealed together via application ofan adhesive.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the devices and methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1. PCL micro- and nano-porous thin-film fabrication for cellencapsulating devices. Panel A) Schematic of the device two-stepheat-sealing and cell encapsulation. Panel B) Cross section SEM of themicro-porous thin-film and (inset) top down image of the film surface.Panel C) Cross-section SEM of the nano-porous thin-film and (inset)top-down image of the nano-porous film surface. Panel D) Image of anassembled device, demonstrating device flexibility.

FIG. 2. In vitro device function. Panel A) In vitro device viability ofencapsulated MIN6 cells as measured with mCherry fluorescence. Panel B)Glucose stimulation index of MIN6 Cells encapsulated in either micro- ornano-porous devices. Panel C) Glucose stimulation of primary isletsencapsulated in micro-porous devices. (p≤0.5, n≥3)

FIG. 3. In vivo device image and tracking. Panel A) Device withencapsulated MIN6 cells implanted in the subcutaneous space of the mousedorsal. Panel B) Device with encapsulated MIN6 cells implanted under themouse skin and muscle over the liver. Panel C) No device control withcells implanted directly into the kidney capsule. Panel D) Device withencapsulated MIN6 cells implanted after 1 day, Panel E) 30 days, PanelF) 90 days.

FIG. 4. Micro-porous barrier inhibits cell invasion. Panel A) Top downSEM image of cells attached to the exterior surface of the micro-porousthin-film device after 1 month in vivo. Panel B) Cross-section SEM imageof the micro-porous thin-film device after 1 month in vivo,demonstrating membrane integrity and isolation of internal and externalcells.

FIG. 5. Cytokine protection. Viability of cells within a micro-porousdevice (Panel A) and a nano-porous device (Panel B) over 1 week, with(solid line) and without (dashed lines) cytokines. (n≥4)

FIG. 6. Device vascularization. Bright field images of devices implantedafter Panel A) 7 days, Panel B) 14 days, Panel C) 30 days and Panel D)90 days, with magnified images at day 7 and day 90. Panel E)Quantification of device vascularization from day 7 to day 90. (n=3)

FIG. 7. Device exterior SEM. Panel A) Device exterior prior toimplantation. Panel B) Device exterior after 2 months in vivo.

FIG. 8. Cytokines affect cell viability. Cell number was quantified by acyquant assay (−) no cytokine control, (+) a combination of cytokineswith TNF alpha, IL1 beta and IF gamma, and TNF alpha, IL1 beta and IFgamma individually.

FIG. 9. Cell-free devices controls for device vascularization. Brightfield images of devices implanted after 50 days Panel A) Porous-PCL,Panel B) Non-porous PCL, Panel C) PLGA, and Panel D) PVDF.

FIG. 10. Histology of devices. Panel A) Cross section of a device after2 months in vivo, with Masson trichrome staining. Panel B) Magnificationof device cross-section, demonstrating minimal fibrotic response.

FIG. 11. Exemplary thin film devices. Panel A) A circular thin filmdevice including an opening for access to the device lumen, the openingincludes a tube for accessing device lumen. Panel B) A rectangulardevice with a thin elongated opening and a tube for accessing devicelumen.

FIG. 12. Graph demonstrating that 20 nm PCL membranes limit diffusion ofIgG.

FIG. 13. Graph demonstrating that human embryonic stem (hES) greenfluorescent (GFP)-insulin cells encapsulated in nano- or macro-porousdevices are viable for up to 5 weeks in vitro.

FIG. 14. Panel A depicts release of c-peptide from islet cellsencapsulated in a device having a polymer layer with about 2 μm diameterpore (200 nm connectivity) or with about 20 nm pore size. Panel Bdepicts glucose stimulation index (GSI) of encapsulated islet cells.

FIG. 15. Panel A) Polypropylene thin film device shows a significantforeign body response (FBR) within 2 weeks after transplantation. PanelB) A PCL thin film device induces minimal FBR 5 months after transplant.

FIG. 16. Exemplary thin film devices showing means for inlet to andoutlet from the devices.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

“Subject” refers to any animal, e.g., a mammal, such as a mouse, rat,goat, dog, pig, monkey, non-human primate, or a human.

“Biocompatible,” as used herein, refers to a property of a material thatallows for prolonged contact with a tissue in a subject without causingtoxicity or significant damage.

As used herein, the terms “treat,” “treatment,” “treating,” and thelike, refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment,” as used herein,covers any treatment of a disease in a subject, particularly in a human,and includes: (a) preventing the disease from occurring in a subjectwhich may be predisposed to the disease but has not yet been diagnosedas having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, e.g., causing regression ofthe disease, e.g., to completely or partially remove symptoms of thedisease.

“Therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result.

“Tubing” or “tube”, as used herein refers to an elongated structurehaving a cylindrical wall defining an interior space. A tubing can havea substantially constant inner diameter along the length of the tubing.The length of the elongated structure may be longer than the width by afactor of 5, 10, 20 or more.

An “end,” as used in reference to an end of a tubing, is meant toindicate an extremity or an extreme portion of the tubing. The end of atubing does not necessarily refer to a physical termination of thetubing, although the end of the tubing may in some cases coincide with aphysical break in the tubing, depending on context.

The “internal volume,” as used in reference to a tubing, refers to thevolume of the space in the tubing bound by the internal wall.

“Lumen” as used in the context of the device of the present disclosurerefers to the space or internal volume created by sealing the first andsecond layers around the edges of the device, wherein the layers aretypically porous.

The terms “layer”, “film”, or “membrane” and plurals thereof as used inthe context of a device of the present disclosure refer to theindividual layers of the device that are typically formed from apolymer. The “layer”, “film”, or “membrane” used to manufacture a porousdevice of the present disclosure is typically porous and can benanoporous or microporous. The phrases “nanoporous layer,” “nanoporelayer,” “nanoporous membrane,” “nanopore membrane,” “nanoporous film,”and “nanopore film” are used interchangeably and all refer to a polymerlayer in which nanopores have been created. A nanoporous layer mayinclude a backing or a supporting layer for structural support. Thebacking may be a microporous layer. The phrases “microporous layer,”“micropore layer,” “microporous membrane,” “micropore membrane,”“microporous film,” and “micropore film” are used interchangeably andall refer to a polymer layer in which micropores have been created.

“Encapsulated” as used in the context of cells disposed in a lumen ofthe devices provided herein refers to cells that are contained withinthe device in the lumen defined between a first and second layer of thedevice.

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the device”includes reference to one or more devices, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

As summarized above, a thin film device for transplantation of apopulation of cells into a subject is provided. Also described hereinare methods for making the device and methods for using such a device.

Device for Transplantation of Cells and Methods of Use

In certain embodiments, a device for transplantation of an encapsulatedpopulation of cells into the body of a subject may include a firstnanoporous or microporous polymer layer, a second nanoporous ormicroporous polymer layer, wherein the first and second layers are incontact with each other along a periphery of the first and second layersthereby defining an enclosed space or a lumen between the first andsecond layers; and a population of cells disposed in the lumen betweenthe first and second layers.

The device is configured to induce minimal foreign body response and topromote vascularization into the device. The porous material of the thinfilm device allows exchange of molecules (e.g., diffusion of oxygen,nutrients, cell metabolism waste products, therapeutic proteins) via thepores but is substantially impermeable to cells and to large proteinssuch as immunoglobulins and limits the transport of smaller proteins,such, as cytokines through the pores. Thus, the devices disclosed hereinisolate the cells present in the lumen of the device from the immunesystem of the subject but allow exchange of smaller molecules supportingthe viability and function of the transplanted cells.

When an implantable device containing cells has been isolated from theimmune system by encasing it in a cell-impermeable layer, theimplantable device often stimulates a local inflammatory response,called the foreign body response (FBR) that has long been recognized aslimiting the function of implanted devices that require solutetransport. FBR has been well described in the literature and is composedof three main layers. The innermost FBR layer, adjacent to an implanteddevice is composed of macrophages and foreign body giant cells. Thesecells form a monolayer of closely opposed cells over the surface of animplanted device. The intermediate FBR layer, lying distal to the firstlayer with respect to the device, is a wide zone (30-100 microns)composed primarily of fibroblasts and fibrous matrix. The outermost FBRlayer is loose connective granular tissue containing new blood vessels.Upon induction of a FBR, an implanted device is isolated from the invivo environment limiting the exchange of molecules with the implanteddevice, limiting the utility of the implanted device as well as, leadingto the death of any cells provided within the implanted device.

The devices disclosed herein do not induce a significant FBR asevidenced by lack of fibrosis around the implanted devices of thepresent disclosure and by the viability of the cells in the device for aprolonged period of time.

The devices disclosed herein support viability of cells present in thelumen of the device upon transplantation into a subject, for at leastone month, at least 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3years, 4 years, 5 years or longer.

The device of the present disclosure is substantially impermeable tocells such that cells do not cross the first and second polymer layersin detectable numbers. The device of the present disclosure issubstantially impermeable to immunoglobulins such that the concentrationof any immunoglobulins that gain access to the lumen of the device isbelow the level needed for an immune response against the cells presentinside the lumen of the device. The device of the present disclosuresubstantially limits cytokines from entering the lumen of the devicesuch that the concentration of cytokines that gain access to the lumenof the device is below the level required for an immune response againstthe cells present inside the lumen of the device.

The devices disclosed herein are typically planar and can have any twodimensional planar shape. In certain embodiments, the device may becircular, elliptical, square, rectangular, or a combination thereof. Insome embodiments, the device may be substantially circular and may havea diameter between 1 cm and 5 cm.

In certain embodiments, the device may include an additional layer forincreasing the rigidity of the device. For example, the device mayinclude a layer of material disposed along a side edge of the device.The additional layer may facilitate maintenance of the device as asubstantially planar device by preventing folding of the device duringplacement in a subject and/or after placement in a subject. The layer ofmaterial may be the same material as used for the first layer or thesecond layer or may be of a different material.

In certain embodiments, the device may be sealed entirely along theedges of the device thereby forming a completely enclosed internal spaceor lumen. In other embodiments, the device may be open at one or morelocations at an edge of the device allowing access to the internal spaceor lumen of the device. When the device includes two openings into thelumen of the device the two openings may be located opposite each other,such as, on opposite sides of the planar device.

In certain embodiments, the device may include a lumen having one ormore openings at which the first and second polymer layers are notsealed together. The device may further include a tubing inserted intothe opening. In certain embodiments, the tubing may be affixed to thedevice by sealing the first and second layers, at the opening, to theexterior wall of the tubing. In certain embodiments, a first end of thetubing may be placed at the opening and positioned in the lumen suchthat a minimal volume of the lumen is taken up by the tubing whileallowing loading of fluids into the lumen of the device. The second endof the tubing, which is distal to the first end may be used as a portfor introducing fluids into the lumen via the tubing.

In certain embodiments, the device may include two openings at which thefirst and second layers are not sealed together thereby defining a lumenwith two openings. Each of the openings may include a tubing placed atthe openings. The first opening may be used as an ingress port forintroducing a fluid (e.g., a cell medium containing cells) into thelumen of the device and the second opening may be used as an egress portfor removing fluids (e.g., fluid containing dead cells, cell debris,etc.) from the lumen of the device. As noted herein, the length of thetubing placed within the lumen may be held at a minimal to maximize thevolume available for the population of cells in the lumen. The tubingmay be affixed to the device by sealing the first and second layers ofthe device to the exterior wall of the tubing at the first ends of eachof the tubings. The second end of the tubing forming one or more accessports into the lumen of the device may be closed when not being used toaccess the interior of the lumen. The second end(s) may be closed byforming a plug at the second end(s). In certain cases, the plug may be asilicone plug.

The length of the tubing may be selected based on a number of factors.For example, a shorter tubing may be used when loading a population ofcells into the lumen of the device ex vivo, for example, prior toplacement of the device into a subject. A longer tubing may be used ifthe tubing is to be used for introducing fluids into and/or removingfluids from the lumen of the device after placement of the device into asubject.

In certain cases, the device may include first and second polymer layersthat are sealed to each other along the edges other than at twolocations creating a lumen defined by the sealed edges and having twoopenings. A first end of a first tubing may be placed at a first of thetwo openings. A first end of a second tubing may be placed at a secondof the two openings. The first ends of the first and second tubing maybe affixed to the device by sealing the first layer and the second layerto the exterior wall of the device, such that the lumen is sealed aroundthe exterior wall of the tubing(s). The second ends of the first andsecond tubing may be connected to a remote fill port which converts thedevice into a refillable, closed system. The remote fill port maycontain a solid detectable backing, which allows for, e.g., percutaneouslocating of the port with a magnet finder if the backing is magnetic.The remote fill port may include two separate chambers in the port foraccess to the second end of the first tubing and the second end of thesecond tubing. In certain cases, the tubing may be, e.g., Silastic®silicone tubing. The port may also contain suture tabs at its base toallow for suturing to soft tissue upon implantation, thus helping resistturning or movement of the port. An exemplary remote fill port isdescribed in WO 2016/028774, which is herein incorporated by referencein its entirety.

In certain embodiments, the device and/or the population of cells in thedevice may be amenable to visualization after placement of the deviceinto the subject. For example, the device and/or cells may befluorescent and as such may be visualized by imaging a portion of thebody of the subject into which the device is placed. A fluorescentdevice may facilitate removal of the device and/or access to the tubingof the device if the device is placed wholly inside a subject.

As noted herein, the device may include a first layer that may benanoporous or microporous and a second layer that may be nanoporous ormicroporous. The microporous and/or the nanoporous layer may be formedas described in U.S. Patent Application Publication No. 20140170204,which is herein incorporated by reference in its entirety.

In certain embodiments, the first and/or the second layer of the devicemay be microporous. The pore diameter in the microporous layer may rangefrom 1 μm to 5 μm, such as, 1 μm to 4.5 μm, 1 μm to 3.5 μm, 1 μm to 2.5μm, 1 μm to 2 μm, 1.5 μm to 3 μm, 1.5 μm to 2.5 μm, 1.5 μm to 3 μm, 1.5μm to 5 μm, e.g., 1 μm, 2 μm, or 3 μm. As explained herein, the pores ina microporous layer may be created by forming a layer that includes apolymer that is water insoluble and a pore forming agent that is awater-soluble polymer. The pore forming agent forms spheres in the waterinsoluble polymer layer which spheres are dispersed along the width ofthe water insoluble polymer layer as well as across the thickness of thewater insoluble polymer layer. The spheres determine the pore diameterand a plurality of spheres when connected across the thickness of thewater insoluble polymer layer (cross-section of the water insolublepolymer layer) provide a continuous channel across the water insolublepolymer layer upon dissolution of the spheres. For the formation of amicroporous layer, the spheres of the water soluble polymer aredissolved by exposing the layer to an aqueous solution, thereby creatingspaces/holes throughout the microporous membrane. The spaces/holes mayhave a diameter of about 1 μm to 5 μm. A series of spaces/holes may bepresent sequentially across the thickness of the water insoluble polymerlayer and interconnected to provide a channel which channel may have avarying diameter. For example, a region in the channel at which twospaces are connected (e.g., where two spheres of the pore forming agentwere in contact with each other and dissolved to provide two spacesconnected to each other) may have a smaller channel diameter than theother regions in the channel. The smallest diameter of the channel maybe referred to as the connectivity diameter. In certain embodiments, theconnectivity diameter may range from 900 nm-100 nm, such as, 900 nm-150nm, 900 nm-200 nm, 700 nm-100 nm, 700 nm-150 nm, 700 nm-200 nm, 500nm-100 nm, 500 nm-150 nm, 300 nm-100 nm, 300 nm-150 nm, or 300 nm-200nm.

In certain embodiments, the first and/or the second layer of the devicemay be nanoporous. The pore diameter in the nanoporous layer may rangefrom 10-300 nm, such as, 20-300 nm, 30-300 nm, 10-200 nm, 20-200 nm,30-200 nm, 10-100 nm, 20-100 nm, 30-100 nm, 30-50 nm, 30-40 nm, 20-250nm, 20-150 nm, or 25-150 nm, e.g., 30 nm, 50 nm, 100 nm, 150 nm, or 300nm. In certain embodiments, the nanopore layer may include a backinglayer to structurally support the nanopore layer. In certainembodiments, the backing layer may be a microporous layer, such as amicroporous layer described herein.

In certain embodiments, the first and/or the second layer of the devicemay have a % porosity of at least 50%, such as, at least 55%, at least60%, at least 65%, or at least 70%, e.g., from 50% to 70%, 55% to 70%,or 55% to 65%. % porosity of the first and second nanoporous polymerlayers of the device may be controlled by the number of nanorods use forthe fabrication of the layer. Similarly, % porosity of the first andsecond microporous polymer layers of the device may be controlled by theamount of pore forming agent (e.g., a water soluble polymer) used duringmanufacture of the microporous polymer layer.

As noted herein, the device is a thin layer device where the individualfirst and second layers of the device are each less than 30 μm inthickness. As such, the first and second layers are less than 30 μmthick, such as, less than 25 μm, 20 μm, 15 μm, 10 μm, or 5 μm, e.g., 20μm-10 μm, 25 μm-5 μm, 20 μm-5 μm, 15 μm-5 μm, 15 μm-10 μm, 13 μm-10 μm,13 μm-8 μm, 12 μm-8 μm, 11 μm-9 μm, 10 μm±5% in thickness.

In certain embodiments, the entire device is less than 60 μm thick andhas a surface area of 0.5 cm² to 5 cm², such as, a surface area largerthan 0.75 cm², larger than 1 cm², larger than 1.5 cm², larger than 2cm², larger than 2.5 cm², larger than 3 cm², larger than 3.5 cm², largerthan 4 cm², larger than 4.5 cm², and up to 5 cm². The device may includea first layer and a second layer. Both layers may be microporous ornanoporous. The diameter of the pores in the microporous polymer layersranges from 1 to 5 μm with a connectivity diameter in the range as notedherein. The diameter of the pores in the nanoporous polymer layer rangesfrom 10 nm to 300 nm, such as, 20-300 nm, 30-300 nm, 10-200 nm, 10-50nm, 20-200 nm, 30-200 nm, 10-100 nm, 20-100 nm, 30-100 nm, 20-250 nm,20-150 nm, or 25-150 nm, e.g., 20 nm, 30 nm, 50 nm, 100 nm, 150 nm, or300 nm.

In certain embodiments, the entire device is less than 50 μm thick andhas a surface area of 0.75 cm² to 4.5 cm² per side. The diameter of thepores in the microporous polymer layers ranges from 1 μm to 3 μm, wherethe connectivity diameter of the pores (diameter of a through channel)may range from 900 nm-100 nm, such as, 900 nm-150 nm, 900 nm-200 nm, 700nm-100 nm, 700 nm-150 nm, 700 nm-200 nm, 500 nm-100 nm, 500 nm-150 nm,300 nm-100 nm, 300 nm-150 nm, or 300 nm-200 nm. The diameter of thepores in the nanoporous polymer layers ranges from 20 nm to 100 nm, suchas, 40-100 nm, 30-50 nm, 20-50 nm, or 25-100 nm, e.g., 20 nm, 30 nm, 50nm, or 100 nm.

In certain embodiments, the entire device is less than 40 μm thick andhas a surface area of 1 cm² to 4 cm² per side. The size of the pores inthe microporous polymer layer ranges from 1 μm to 3 μm with aconnectivity diameter in the range as noted herein. The size of thepores in the nanoporous polymer layers ranges from 25 nm to 100 nm, suchas, 25-75 nm, 30-50 nm, 25-50 nm, or 25-40 nm, e.g., 25 nm, 30 nm, 50nm, or 100 nm.

In certain embodiments, the entire device is less than 30 μm thick andhas a surface area of 1 cm² to 2 cm² per side. The diameter of the poresin the microporous polymer layer ranges from 1 μm to 3 μm with aconnectivity diameter in the range as noted herein. The diameter of thepores in the nanoporous polymer layers ranges from 30 nm to 100 nm, suchas, 30-75 nm, 30-50 nm, 40-100 nm, 40-75 nm, 40-50 nm, 50-100 nm, or50-75 nm, e.g., 30 nm, 40 nm, 50 nm, 60 nm, or 80 nm. For example, thefirst and second layers may each be 10 μm±3 μm thick. In certainembodiments, the first layer may be microporous and the second layer maybe nanoporous or vice versa.

In certain embodiments, the device includes a first layer and a secondlayer, each of which are each 10 μm±3 μm thick, wherein the entiredevice has a surface area of 1 cm² to 2 cm² per side. The first layerand the second layer are nanoporous and diameter of the pores in thenanoporous polymer layers ranges from 30 nm to 100 nm, such as, 30-75nm, 30-50 nm, 40-100 nm, 40-75 nm, 40-50 nm, 50-100 nm, or 50-75 nm,e.g., 30 nm, 40 nm, 50 nm, 60 nm, or 80 nm. In other embodiments, firstlayer and the second layer are microporous and the diameter of the poresin the microporous polymer layer ranges from 1 μm to 3 μm (e.g., 2μm±0.5 μm diameter) with a connectivity diameter ranging from 900 nm-100nm, such as, 900 nm-150 nm, 900 nm-200 nm, 700 nm-100 nm, 700 nm-150 nm,700 nm-200 nm, 500 nm-100 nm, 500 nm-150 nm, 300 nm-100 nm, 300 nm-150nm, or 300 nm-200 nm.

The devices of the present disclosure are configured to prevent immunecells from entering the lumen of the device and substantially inhibitantibodies and cytokines from entering the lumen while promotingnutrient exchange with the encapsulated cells and release of therapeuticproteins secreted by the encapsulated cells as well as diffusion ofmetabolic waste products out of the device. The pores and the thicknessof the thin layers forming the thin film device of the presentdisclosure are configured to permit passage of small molecules, such assalts, sugars, amino acids, dopamine, glucose, insulin and substantiallyinhibit passage of large molecules such as, antibodies, C3b, cytokines(e.g., interferons, interleukins, tumor necrosis factors, and the like)and of cells. The first and second porous polymer layers are configuredto allow exchange of small molecules that have a molecular weight lessthan 10 kDa and/or a hydrodynamic radius of less than 2 nm. The firstand second porous polymer layers are configured to substantially limitlarge molecules that have a molecular weight greater than 15 kDa and/ora hydrodynamic radius of greater than 2 nm from crossing the layers andentering the lumen of the device.

Any polymer material may be used to form the first and second porouslayers of the devices disclosed herein. Representative polymers includemethacrylate polymers, polyethylene-imine and dextran sulfate,poly(vinylsiloxane) ecopolymerepolyethyleneimine, phosphorylcholine,poly(ethyl methacrylate), polyurethane, poly(ethylene glycol),poly(lactic-glycolic acid), hydroxyapetite, poly(lactic acid),polyhydroxyvalerte and copolymers, polyhydroxybutyrate and copolymers,polycaprolactone, polydiaxanone, polyanhydrides, polycyanocrylates,poly(amino acids), poly(orthoesters), polyesters, collagen, gelatin,cellulose polymers, chitosans, and alginates or combinations thereof.Additional examples that may be used to coat the scaffold include butare not limited to: collagen, fibronectin, extracellular matrixproteins, vinculin, agar, and agarose. It should be understood thatvarious mixture of the polymers may be used.

In certain embodiments, the first layer and/or the second layer may beformed from poly(caprolactone) (PCL). The PCL used for forming the firstand/or the second layer may have a number average molecular weight (Mn)higher than 50 kDa, such as, higher than 55 kDa, 60 kDa, 65 kDa, or 70kDa, and up to 200 kDa, e.g., 50-200 kDa, 55-200 kDa, 60-200 kDa, 65-200kDa, 70-200 kDa, 50-150 kDa, 55-150 kDa, 60-150 kDa, 65-150 kDa, 70-150kDa, 50-100 kDa, 55-100 kDa, 60-100 kDa, 65-100 kDa, 70-100 kDa, 50-90kDa, 55-90 kDa, 60-90 kDa, 65-90 kDa, or 70-90 kDa. Furthermore, themolecular weight of the PCL polymer may be selected based on theduration for which the device is to be maintained in the body of asubject without substantial degradation of the polymer.

Under physiological conditions, a biodegradable polymer such as PCLpolymer degrades by random chain scission, which gives rise to atwo-phase degradation. Initially, as molecular weight decreases thephysical structure is not significantly affected. Degradation takesplace throughout the polymer material, and proceeds until a criticalmolecular weight is reached, when degradation products become smallenough to be solubilized. At this point, the structure starts to becomesignificantly more porous and hydrated. In certain cases, a highermolecular weight polymer may be used when the device is to be used forproviding transplanted cells into the subject for at least one year. Forexample, when the device is to maintain the integrity of the porouspolymer layers for at least one year, the porous polymer layers may bemade from a polymer, that has at least 70 kDa Mn, e.g., at least 75 kDaMn, at least 80 kDa Mn, or at least 85 kDa Mn, or at least 90 kDa Mn,and up to 100 kDa Mn. In embodiments where the device is configured todegrade by 6 months, the first and second porous polymer layers may beformed from a lower molecular weight polymer, such as, a polymer havingMn of 10-20 kDa.

In some embodiments, the biodegradable polymer includes a blend ofpolymers where the polymers may be of the same or a different type ofpolymer, and each polymer may be of a different MW. In some embodiments,the biodegradable polymer includes a blend of a high MW polymer and alow MW polymer. The high MW polymer may be of about 25 kDa or more, suchas about 30 kDa or more, about 40 kDa or more, about 50 kDa or more,about 60 kDa or more, about 70 kDa or more, about 80 kDa or more, about90 kDa or more, or about 100 kDa, and up to 150 kDa. The low MW polymermay be of about 20 kDa or less, such as about 15 kDa or less, about 10kDa or less, about 8 kDa or less, about 6 kDa or less, and down to 4kDa.

In some embodiments, the ratio by mass of the high MW polymer to the lowMW polymer in a blend of polymers is between about 1:9 and about 9:1,such as between about 2:8 and about 8:2, between about 2:8 and about6:4, or between about 2:8 and about 1:1. In certain embodiments, theratio by mass of the high MW polymer to the low MW polymer is about3:17, about 2:8, about 1:3, about 3:7, about 7:13, about 2:3, about9:11, about 1:1, about 11:9, or about 3:2.

In certain embodiments, thin film devices for transplantation of cellsinto the body of a subject do not comprise poly(lactic-co-glycolic acid)(PLGA), polyvinylidene difluoride (PVDF), alginate, collagen, gelatin,agarose, silicon, cellulose phosphate, or polyproplylene (PP).

In certain embodiments, the exterior surface of the device may bemodified by disposing one or more agents that improve the device. Forexample, molecule that promotes vascularization of the device orinhibits immune or inflammatory response to the device may be disposedon the exterior of the device. Such molecules include, but are notlimited to VEGF (vascular endothelial growth factor), PDGF(platelet-derived growth factor), FGF-1 (fibroblast growth factor),angiopoietin MCP-1, αvβ3, αvβ5, CD-31, VE-cadherin, ephrin, plasminogenactivators, angiogenin, Del-1, aFGF (acid fibroblast growth factor),vFGF (basic fibroblast growth factor), follistatin, G-CSF (granulocytecolony-stimulating factor), HGF (hepatocyte growth factor), Leptin,placental growth factor, PD-ECGF (platelet-derived endothelial growthfactor), and the like.

The devices of the present disclosure are used to provide encapsulatedcells in a subject. The method for providing the encapsulated cellsincludes providing a thin film device, e.g., a multilayer thin filmdevice, as provided herein, transplanting the thin film device into thesubject, promoting vascularization into the lumen of the device via thepores in the first polymer layer and/or second polymer layer; limitingforeign body response to the device; limiting ingress of cells,immunoglobulins, and cytokines into the lumen via the first and thesecond polymer layers; and releasing from the first polymer layer and/orthe second polymer layer molecules secreted by the population of cells.The devices disclosed herein may promote vascularization into the lumenof the device such that at least 20% of the device is vascularizedwithin a month after transplantation into a subject, such as at least a30% vascularization, 40% vascularization, 50% vascularization, 60%vascularization within a month after transplantation into a subject. Thedevices disclosed herein may limit the diffusion of cytokines andimmunoglobulins through the pores in the first polymer layer and secondpolymer layer such that the diffusion rate is less than 50%, less than40%, less than 30%, less than 20% compared to diffusion in absence of abarrier layer.

The devices of the present disclosure are sized to house an effectivenumber for transplanted cells for treatment of a subject in needthereof. For example, the subject may be suffering from a conditioncaused by lack of functional cells, e.g., wherein molecules typicallysecreted by functional cells are not secreted or are secreted at a levelresulting in the condition. Providing functional cells could alleviatethe condition. Exemplary conditions include type 1 diabetes, Parkinson'sdisease, muscular dystrophy and the like.

The device may be transplanted into any suitable location in the body,such as, subcutaneously, intraperitoneally, or in the brain, spinalcord, pancreas, liver, uterus, skin, bladder, kidney, muscle and thelike. The site of implantation may be selected based on thediseased/injured tissue that requires treatment. For treatment of adisease such as diabetes mellitus (DM), the device may be placed in aclinically convenient site such as the subcutaneous space or theomentum.

Populations of cells for transplantation using the devices describedherein include but are not limited to, bone marrow cells; mesenchymalstem cells, stromal cells, pluripotent stem cells (e.g., inducedpluripotent stem cells and embryonic stem cells), blood vessel cells,precursor cells derived from adipose tissue, bone marrow derivedprogenitor cells, intestinal cells, islets, Sertoli cells, beta cells,progenitors of islets, progenitors of beta cells, peripheral bloodprogenitor cells, stem cells isolated from adult tissue, retinalprogenitor cells, cardiac progenitor cells, osteoprogenitor cells,neuronal progenitor cells, and genetically transformed cells, or acombination thereof. The encapsulated cells may be from the subject(autologous cells), from another donor (allogeneic cells) or from otherspecies (xenogeneic cells). The cells can be introduced into the lumenof the device and the device may be immediately (within a day) implantedinto a subject or the cells may cultured for longer period, e.g. greaterthan one day, to allow for cell proliferation prior to implantation. Thenumber of cells introduced into the lumen of the device may vary and maybe determined empirically.

In certain embodiments, the devices disclosed herein may be used totreat a person having diabetes, such as, type 1 diabetes. The device mayinclude pancreatic islet cells or may include stem cells that arecapable of differentiating into pancreatic islet cells. In certainembodiments, pluripotent stem cells (PSCs) may be differentiated intopancreatic islet cells inside the device and then the device containingthe differentiated pancreatic islet cells is placed in the subject(e.g., in the omentum, adjacent to pancreas or liver). In some case, thedevice may include PSCs and the device may be implanted adjacent thepancreas or liver of the subject.

As noted herein, the devices disclosed herein may maintain thetransplanted cells in a functional and viable state for at least 1 monthand up to a period of at least 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to ayear or longer. In certain cases, the integrity of the device ismaintained for at least one year and the cells in the device may bereplaced with a fresh population of cells using the tubing attached tothe device (e.g., via a remote fill port) while the device is locatedinside a subject.

The methods and devices disclosed herein can be used for both humanclinical and veterinary applications. Thus, the subject or patient towhom the device is administered can be a human or, in the case ofveterinary applications, can be a laboratory, agricultural, domestic, orwild animal. The subject devices and methods can be applied to animalsincluding, but not limited to, humans, laboratory animals such asmonkeys and chimpanzees, domestic animals such as dogs and cats,agricultural animals such as cows, horses, pigs, sheep, goats, and wildanimals in captivity such as bears, pandas, lions, tigers, leopards,elephants, zebras, giraffes, gorillas, dolphins, and whales.

Methods of Preparation

Also provided are methods of preparing the subject devices. In someembodiments, the method includes obtaining or fabricating a firstmicroporous or nanoporous polymer layer and a second microporous ornanoporous polymer layer, where the first and second layers aresimilarly sized. The first layer has a first surface and a secondsurface opposite the first surface. The second layer also has a firstsurface and a second surface opposite the first surface. The methodfurther comprises positioning the second layer over the first layer suchthat the edges of the layers are aligned and the second surface of thesecond layer is in facing configuration with the first surface of thefirst layer. The method further comprises sealing the second surface ofthe second layer to the first surface of the first layer. The seal maybe formed along a side edge of the first and second layers forming anenclosed space or lumen in between the second surface of the secondlayer and the first surface of the first layer. The seal may initiallybe formed along the side edges of the first and second polymer layerswhile leaving an unsealed edge. A population of cells may introducedinto the lumen via the opening and the opening may then be closed bysealing the second surface of the second layer to the first surface ofthe first layer, thereby enclosing the population of cells inside thedevice.

Any method for obtaining or fabricating a microporous or nanoporouspolymer layer may be used. For example, a microporous polymer layer anda nanoporous polymer layer may be fabricated using a method disclosed inUS Patent Application Publication 20140170204, Steedman et al.,Biomedical Microdevices, 12(3) (2010) 363-369, Bernards et al., J. Ocul.Pharmacol. Ther. 2013, 29, 249-257; Bernards et al., Adv. Mater. 2010,22, 2358-2362; or Bernards et al., Nano Lett. 2012, 12, 5355-5361, eachof which is herein incorporated by reference in its entirety.

In certain embodiments, the population of cells may be loaded into thelumen via an opening. In other embodiments, a tubing may be insertedinto the opening and used to load the population of cells into thelumen. In certain cases, the tubing may be retracted from the openingand the opening sealed after the population of cells has been introducedinto the lumen.

In certain embodiments, the tubing inserted into the opening may beaffixed to the device by sealing the second surface of the second layerand the first surface of the first layer to the exterior wall of thetube.

In certain embodiments, the seal may initially be formed along the sideedges of the first and second polymer layers while leaving unsealededges at two locations, thereby providing a first and a second openinginto the lumen. A first end of a first tubing may be inserted into thefirst opening and the tubing fixed in place by sealing the first andsecond layers to the exterior wall of the tubing. A first end of asecond tubing may be inserted into the second opening and the tubingfixed in place by sealing the first and second layers to the exteriorwall of the tubing. The second ends of each tubing may be configured forattachment to a remote fill port for forming a refillable, closedsystem, e.g., after the in vivo placement of the device, the second endof each the tubing may be attached to a remote fill port, such as aremote fill port described in WO 2016/028774.

Sealing of the first and second layers to each other or to a tubing maybe carried out using an adhesive, or by using heat, or a solvent to meltthe layers. Sealing of the two layers may be at the outermost edge ofthe layers or at a location adjacent to the outermost edge of thelayers.

In some embodiments, the device may include a third polymer layerdisposed along the edges of the device to provide a thicker edge to thedevice. This third polymer layer may be disposed along the entire edgeof the device or on a portion thereof. The portion of the edge of thedevice to be covered by the third polymer layer may be determined by thein vivo location at which the device is to be disposed. The third layermay be affixed to the edge of the device via use of an adhesive, heat,or a solvent to melt the layers together. The third layer may be anonporous polymer layer or a microporous polymer layer. The third layermay have a thickness of at least 10 μm, such as, at least 30 μm, 100 μm,300 μm, 500 μm, 1 mm, or 2 mm or higher, e.g., 100 μm-2 mm, 300 μm-2 mm,500 μm-2 mm, or 1 mm-2 mm.

In embodiments, where nanoporous polymer layers are used to fabricatethe device, the nanoporous layer may include a microporous backinglayer. The microporous polymer layer used for structurally supportingthe nanoporous polymer layer may be made from the same polymer as thenanoporous layer. The microporous polymer layer is applied to thenanoporous layer such that the two layers are in contact with each otheracross a surface of the two layers. The nanoporous layer with themicroporous backing may be used in either orientation when forming thedevice.

In certain embodiments, the sealing is performed using an annulus thatmay be heated. An exemplary method includes the use of U-shaped wirethat is heated to a temperature (e.g., 80° C.) above the meltingtemperature of the polymers (e.g., PCL) used in the fabrication of thedevice. The wire may be placed underneath the two layers and heatedthereby melting the two layers at the locations adjacent the wire. Insome examples, the first and second porous polymer layer may be placedover a U-shaped nichrome wire embedded in PDMS (Sylgard 184). Thediameter of the wire may be selected based on the desired volume of thelumen. To secure the first and second layers a weight (e.g., PDMSweight) may be placed over the first and second layers holding themflat. A current may then be applied to the wire for melting the layersand defining the lumen shape and leaving an opening. The size and shapeof the wire may be selected to produce devices of a desired size.Subsequent to loading of the cells into the lumen the open side of thedevice may be closed.

In certain embodiments, the sealing step of the subject methods isperformed using a laser beam to heat a defined area of the thin filmlayers, for example, a circular area surrounding the area where thecells are to disposed. In certain embodiments, the sealing step of thesubject methods is performed by disposing an adhesive material on one orboth of the first and second layers. For example, an adhesive materialmay be disposed on the first layer and/or the second layer in an areasurrounding the area where the lumen is to be created. The adhesive mayseal the two layers when the two layers are brought in contact.Alternatively, the adhesive may be a heat sensitive adhesive or apressure sensitive adhesive. In these embodiments, heat or pressure maybe applied in order to seal the layers of the thin film device. It willbe understood by those of skill in the art that any method effective toseal the first and second layers to each other or to a tubing may beused in making the devices described herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Materials and Methods:

Chemicals were purchased from Sigma Aldrich unless noted and cellculture materials were purchased from the University of California, SanFrancisco (UCSF) cell culture facility. All films were spuncast ontosilicon wafers at 1000 rpm for 30 seconds followed by 2000 rpm for 30seconds. Devices were characterized with a Carl Zeiss Ultra 55Field-Emission Scanning Electron Microscope using an in-lens SecondaryElectron detector.

Micro-Porous Thin-Film Fabrication

Micro-porous PCL thin-films were spuncast from a solution of 150 mg/mLPCL (70-90 kDa Mn) and polyethylene glycol (PEG, 2 kDa Mn) in2,2,2-trifluoroethanol, which was prepared by stirring at 65° C. untildissolved. Following spin-casting, the PEG was dissolved by soaking inwater for 1 hour, resulting in micro-porous PCL films with poresapproximately 2 μm in diameter. Devices were 1 cm in diameter resultingin a surface area of 1.57 cm² per side, with 67.5±1.3% porosity and0.37±0.02 density.

Nano-Porous Thin-Film Fabrication

Nano-porous PCL films were formed using an established template-basedapproach reported elsewhere (Bernards, D. A. and Desai, T. A., Adv.Mater. 2010, 22, 2358-2362). In brief, a 0.5 M solution of zinc acetatedihydrate and ethanolamine in 2-methoxyethanol was spuncast onto siliconwafers and annealed at 300° C. on a hot plate to generate a zinc oxide(ZnO) seed layer. From this seed layer, ZnO nanorods were hydrothermallygrown in a 5 mM zinc acetate solution at 85-90° C. for two hours. A 150mg/mL PCL solution was then spuncast onto the nanorods followed by a 150mg/mL PEG:PCL solution to provide a micro-porous support, creating anano-porous film with a micro-porous backing support layer. The film wassoaked in a dilute sulfuric acid solution to etch away the ZnO nanorodsand also dissolve the PEG, resulting in a nano-porous membrane withpores ranging from 30 nm to 100 nm supported by a micro-porous backing.Membrane characterizations and ZnO nanorod morphology were previouslymeasured (Bernards et al., J. Ocul. Pharmacol. Ther. 2013, 29, 249-257;Bernards et al., Adv. Mater. 2010, 22, 2358-2362; Bernards et al., NanoLett. 2012, 12, 5355-5361).

Non-Porous Membrane Fabrication

Non-porous PCL films were spuncast from a solution of 150 mg/mL PCL(70-80 kDa Mn) in 2,2,2-trifluoroethanol, which was prepared by stirringat 65° C. until dissolved. Non-porous poly(lactic-co-glycolic acid)(PLGA) films were spun cast from a solution of 300 mg/mL PLGA (85:15LA:GA 45 kDa Mn) in 2,2,2-trifluoroethanol. Polyvinylidene fluoride(PVDF) film was prefabricated from Sigma and cut to shape.

Assembly of Thin-Film Devices

Devices consisted of two PCL thin-films heat-sealed together usingresistive heating of a nichrome wire. A two-step heat-sealing method wasused where 1.2 Amp current ran through a nichrome wire outlining theregions to be sealed. For the first sealing step, two films were placedover a U-shaped nichrome wire embedded in PDMS (Sylgard 184), 1 cm indiameter. To secure the membranes a PDMS weight was placed over thefilms, holding them flat. A 1.2 Amp current ran through the wire for 30seconds and sealed the devices in the shape of a U, defining the devicelumen shape and leaving an open side for cell injection. 1.5 MillionMIN6 cells in high glucose Dulbecco's Modified Eagle's (DME) wereinjected into the devices through the remaining open side. Second, theremaining side of the device was sealed by placing the open edge over astraight nichrome wire embedded in PDMS and heat-sealed with a 1.2 Ampcurrent for 30 seconds.

Characterization Using Scanning Electron Microscopy of Films and Devices

Micro- and nano-porous thin PCL films were mounted on a flat SEM mountwith colloidal graphite (Ted Pella, Inc.). Cross sections wereflash-dipped in isopropanol followed by liquid nitrogen freeze fractureand then mounted. Devices from in vivo experiments were fixed with 3.7%formaldehyde for 30 minutes, washed in deionized water three times, thensequentially dehydrated in increasing ethanol concentrations andmounted.

Cell Culture

MIN6 cells were cultured using standard media conditions (Miyazaki, etal., Endocrinology 1990, 127). Genes for mCherry and puromycinresistance were introduced using a lentivirus construct designed by theLentiviral Core at UCSF. The cells were transduced using standardprotocol with a multiplicity of infection of 2, and transduced cellswere selected using puromycin. Genes encoding firefly luciferase andgreen fluorescence protein were similarly introduced into MIN6 cells.Primary islets were isolated by the Islet Core at UCSF using standardislet isolation protocols (Szot et al., J. Vis. Exp. 2007, 1640, 255).

Glucose Stimulated Insulin Secretion

Insulin secretion was analyzed using a glucose-stimulated insulinsecretion assay. Cells were rested for 30 minutes in medium-containing 5mM glucose and then stimulated using medium-containing 15 mM glucose.Culture supernatant was collected at 30 minutes and 60 minutes afteraddition of high glucose. Insulin protein content in the culturesupernatant was measured using an enzyme linked immunosorbant assay(Mercodia). The ratio of insulin secreted at high to low glucoseconditions was used to calculate the glucose stimulation index.

Cytokine Assay

To determine the effect of cytokines on the viability of encapsulatedbeta cells, 250,000 cells in micro- or nano-porous devices were culturedin a cytokine cocktail consisting of TNFα (300 ng/mL; VWR), IL1β (110ng/mL; VWR) and IFNγ (200 ng/mL; Fisher) in high glucose DME media, with10% fetal bovine serum, 1% penicillin, and 1% streptomycin. The deviceswere imaged daily for the mCherry signal using a standardspectrophotometer. The signal intensity was measured for each respectivedevice for 7 days, and normalized against the initial signal.

Bioluminescent Imaging

Thin-film devices with luciferase-expressing MIN6 (MIN6.LUC) cells wereimplanted, in either the subcutaneous space on the dorsal aspect, theabdominal cavity between the muscle wall and the liver of MOD.Cg-Prkdc^(scid) IIl2rg^(tm1Wjl)/SxJ (NSG) or BALB/C mice. Persistence ofthe encapsulated cells in vivo was assessed by monitoring luciferaseactivity using a Xenogene IVIS 200 imaging system (Perkin Elmer). Theanimals transplanted with MIN6.LUC cells, were injected IP withD-luciferin solution (Goldbio, St. Louis, Mo.) at the dose of 150 mg/kg8 minutes before imaging in order to capture the peak in bioluminescentintensity, as previously described (Fowler et al., Transplantation 2005,79, 768-776). The mice were anesthetized with an isoflurane mixture (2%in 98% O₂) and imaged by using a Xenogen IVIS 200 imaging system.Bioluminescent images were acquired for 1 minute and then analyzed usingthe Living Image analysis software (Xenogen Corp., Alameda, Calif.).Regions of interests (ROI) were centered over the bioluminescentregions. Photons were counted within the ROI over the acquisition time.Adherence to the same imaging protocol ensured consistent signaldetection and allowed us to compare data acquired over a period of atleast 3 months.

Histology

Mouse tissue samples were collected and fixed in 4% paraformaldehyde for24 hours, washed with phosphate buffered saline at 4° C. for 48 hours,then 30% sucrose for 24 hours. Samples were then taken to the MousePathology Core at UCSF and Optimal Cutting Temperature (OCT) embedded,sliced, and Hematoxylin- and Eosin-stained or Masson's-Trichrome-stainedby either the Mouse Pathology Core or the Histology and Imaging Core atUCSF.

Vasculature

At 7, 14, 30, and 90 days after transplantation, PCL device-bearing micewere anesthetized with an intraperitoneal injection of Avertin solution2.5% (Sigma) and subjected to optical imaging using a Leica MZ16Fmicroscope (Leica Biosystems, Wetzlar, Germany). The animals wereeuthanized by cervical dislocation and the encapsulated devices werecollected for further analysis. The images of the encapsulated graftswere analyzed using ImageJ software. Vessel density was measured byautomated counting of red pixels divided the area of the ROI within thedevice; a threshold was previously set for the red channel to subtractbackground.

The present disclosure describes fabricated and characterizedpoly-caprolactone (PCL) thin-film macroencapsulation devices as aninnovative strategy to address the challenges of existing micro- andmacroencapsulation approaches. The thin compliant design allowsdiffusion and flexibility similar to microencapsulation approaches,while the device surface area allows precise membrane control andretrievability, features associated with larger macroencapsulationtechnologies. The studies described herein show that encapsulated cellsdemonstrated viability, function, protection from immune-cell intrusion,protection from cytokine-mediated cell death, and neovascularization.PCL has been used in FDA-approved medical devices and has demonstratedlong-term biocompatibility in multiple animal models (Bernhardt et al.,Biomatter. 2012, 2, 158-265; Bernards, D. A. and Desai, T. A. SoftMater. 2011, 6, 1621-1631; Bernards et al. J Ocul Pharmacol Ther. 2013,29, 249-257; Bernards, D. A. and Desai T. A. Rev Adv Mater Sci. 2010,22, 2358-2362; Abedalwafa et al. Rev Adv Mater Sci. 2013, 34, 123-140;Angius et al., Biomaterials 2013, 33, 8034-8039). Additionally, PCLdegradation can be tuned to match the lifetime of the encapsulatedcells, eliminating the need for device removal (Bernards, et al., NanoLett. 2012, 12, 5355-5361; Mendelsohn et al, Langmuir. 2010, 26,9943-9949). The use of porous PCL thin films allows a thin and flexibledevice to be designed with either micro- or nanoscaled features leadingto better nutrient exchange, precise membrane control, and devicetracking. In this study, the MIN6 cell line, a well-established mouseinsulinoma cell line known to respond to glucose with insulin secretion,was used as a model for islet beta cells. Using MIN6 cells provides asustainable and consistent source of cells across experiments. Primaryislets were also used to demonstrate long-term viability of encapsulatedcells.

We describe the fabrication of micro-porous and nano-porous PCLthin-film cell-encapsulation devices, cell behavior in these devices,and in vivo integration of these devices in allogeneic mouse models. Todesign these encapsulation devices, the geometry was engineered tocombine the advantages of the precise membrane control ofmacro-encapsulation devices with improved nutrient exchange ofmicro-encapsulation devices. Furthermore, the choice of PCL was based onits range of molecular weights, tunable degradation profile,flexibility, and use as a non-toxic material in FDA-approved medicaldevices. Two different methods were used to create micro- andnano-porous membranes for thin-film devices. The micro-porous filmsutilize phase separation of PEG and PCL in solution. In this method,after films are cast, the pore forming agent (PEG) is dissolved, leavinga micro-porous film (Bernards et al., Nano Lett. 2012, 12, 5355-5361).By tuning the concentration of ratio and composition of the two polymersfilms can be tailored for a variety of porosities and architectures(Bernards et al., Soft Mater. 2011, 6, 1621-1631; Bernards et al., NanoLett. 2012, 12, 5355-5361; Lu et al., Int. J. Pharm. 2011, 419, 77-84;Lin et al., J. Control. Release 2003, 89, 179-187; Rong et al., Int. J.Pharm. 2012, 427, 242-251; Anzai et al., Colloids Surfaces BBiointerfaces 2015, 127, 292-299; Lei et al., Eur. J. Pharm. Biopharm.2011, 78, 49-57; Online, V. A.; Ledeuil, J. B.; Uhart, A.; Allouche, J.;Dupin, J. C.; Martinez, H. New Insights into Micro/Nanoscale Combined.2014, 11130-11140). Nano-porous films were created from a zinc oxidenanorod template and backed with a micro-porous support layer. Zincoxide nanorod dimensions can be readily tuned, allowing a wide range ofpores sizes and giving the ability to further refine these devices (Kimet al., Nanotechnology 2014, 25, 135609; Zhang et al., Chemistry 2005,11, 3149-3154).

FIG. 1, Panel A schematically details the method for heat-sealing twothin-films to generate a single device. Two-step sealing decouplesdevice shape from cell encapsulation. A first heat-sealing step controlsthe device size. Once the device outline is sealed, cells are insertedinto the lumen of the thin-film device, and a second heat-sealing stepencapsulates the cells. Device geometry can be arbitrarily selectedbased on the shape of the nichrome wire that defines the device seal,typically from 1 cm to 5 cm in diameter, allowing devices to be scaledto contain more cells as necessary.

Scanning electron microscopy (SEM) was used to visualize themicro-porous thin films, which had approximately 2 μm sized pores and amembrane thickness of approximately 10 μm (FIG. 1, panel B). Similarly,a SEM image cross-section and top-down image of a nano-porous thin-filmwith a micro-porous backing showed a membrane thickness of 10 μm andnano-pores ranging from 30 nm-100 nm (FIG. 1, Panel C). The thin design,flexibility, compliance of the material, and structure of the device asa whole creates a cell-encapsulating device that is easy to handle withprecise membrane control (FIG. 1, Panel D). Noting that oxygen diffusionin aqueous solutions is 100 μm to 200 μm, these thin-film devices withmembrane thicknesses of 10 μm decrease the proximity to vasculatureneeded for adequate oxygen consumption (Wendt, et al., Adv. Mater. 2009,21, 3352-3367; Martin et al., Biomaterials 2005, 26, 7481-7503). Giventhe thin-film nature of the devices, the total cell content scales withdevice area, while the average distance of cells from the nutrientsource at the device exterior is maintained, bridging the advantages ofboth micro- and macro-encapsulation technologies. The thin-film designof the device, coupled with rapid device vascularization will providesufficient oxygen for encapsulated cells.

mCherry-expressing MIN6 cells encapsulated in either micro- ornano-porous devices maintain viability in vitro through 6 days, asdefined by the persistence in mCherry signal, and are able to maintainglucose stimulated insulin secretion (FIG. 2, Panel A). The glucosestimulation index is a metric to quantify beta cell function bycomparing the ratio of insulin release in a high glucose state relativeto a resting state. MIN6 cells encapsulated in either micro- ornano-devices demonstrate no statistically significant changes in theirglucose stimulation index (FIG. 2, Panel B). Furthermore, freshlyisolated mouse islets encapsulated in these devices maintain theirglucose stimulation index over a period of 20 days in vitro, which issignificantly improved over free islets alone, which have over a 25%decrease in the glucose stimulation index from day 1 (FIG. 2, Panel C).This demonstrates that beta cell insulin response to glucose ismaintained within both nano- and micro-porous thin-film devices.Furthermore, glucose sensing and insulin secretion, a major function ofbeta cells, is unaffected by encapsulation in either micro- ornano-devices.

Viability and persistence of transplanted cells can be monitored inrecipient mice in real time using bioluminescence imaging. Thistechnique was used to monitor in vivo luciferase-expressing MIN6(MIN6.LUC) cells encapsulated into thin-film devices implanted under theabdomen above the liver (FIG. 3, Panel A) or over the muscle layer inthe subcutaneous space of the mouse dorsal flank (FIG. 3, Panel B) orunencapsulated cells implanted into the kidney capsule (FIG. 3C) ofsyngeneic B6 mice. The thin-film device design is compatible withbioluminescence imaging and thus allows tracking of encapsulated cellsin vivo. Luciferase-expressing MIN6.LUC cells were encapsulated intothin-film devices and the devices were implanted under the abdomen abovethe liver or over the muscle layer in the subcutaneous space of themouse dorsal flank (FIG. 3, Panels D-F). Bioluminescent signal decreaseswith device implant depth, and both implanted device locations werevisually brighter than the no device kidney capsule control. Persistenceof bioluminescent signal demonstrates maintained viability through 90days of implantation. As the bioluminescent signal tracks with devicelocation, it also provides a non-invasive method to track devicemovement. Because the encapsulated cells are not fixed within thedevice, and the device itself is not sutured or tethered to any tissue,cellular reorganization of the encapsulated cells or daily movement ofthe mouse can result in the movement of the bioluminescent signal.

Ideal immune protection requires physically excluding immune cells aswell as restricting diffusion of immune mediators such as cytokines thatare toxic to beta cells. By encapsulating cells in micro-porous devices,cell-contact-mediated immune protection may be achieved, and additionalcytokine-mediated immune protection may be accomplished with thenano-porous devices. Cells encapsulated in thin-film devices arephysically compartmentalized from the in vivo environment, as clearlyseen in FIG. 4, Panel A, where cells are attached to the outer surfaceof the device but no infiltration into the device lumen was found.Despite cell adhesion on device surfaces, pores remain unclogged (FIG.7) most likely due to the limited fibrotic response of the surroundingtissue. FIG. 4, Panel B shows a SEM cross-section, with a cell attachedto the external surface of a device. No cellular processes are seenextending into the device, further confirming the ability of the deviceto prevent cell-contact-mediated interaction by isolating theencapsulated cells from the surrounding in vivo tissue. By furthercontrolling the porosity of the membrane, cytokine-mediated immuneprotection may additionally be achieved. Tumor necrosis factor α (TNFα),interleukin 1 β (IL1β), and interferon γ (IFNγ) inflammatory cytokinesare known to kill beta cells individually, and act synergistically whenpresent in combination. They were chosen in order to test the devices'ability to protect from cytotoxic cytokines (Tracey, K. J. TheInflammatory Reflex. 2002, 420, 853-859; Bastiaens, P. When It Is Timeto Die. 2009, 459; Libert, C. A Nervous Connection. 2003, 421, 328-329;Wang et al., Cell 2008, 133, 693-703). (FIG. 8) (Fonseca et al., Int.Immunopharmacol. 2014; Roff et al., Front. Immunol. 2014, 4, 498; Yanget al., Mol. Endocrinol. 2014, me20131257). Interestingly, whereasmicro-porous thin-film devices failed to maintain cell viability (FIG.5, Panel A), the use of a nano-porous layer in these thin-film devicesmitigated the cytokine-mediated decrease in viability (FIG. 5, Panel B).It is unclear if cytokines are completely isolated from the lumen ofdevices, given the size of cytokines in relation to the nano-pores, aportion of cytokines are expected to pass through the membrane. Theprotection by nano-porous devices would result from limited transportand diffusion of cytokines though the membrane, such that the cells areunresponsive to the reduced cytokine concentrations. Considering thatthe cytokine cocktail concentration used exceeds known cytotoxicconcentrations by 10-fold, we expect that the majority of the cytokinesto be limited by the nano-porous barrier. This further highlights howmicro-porous and nano-porous membranes can be used to control desiredcell responses.

Device vascularization in vivo is imperative for long-term survival ofencapsulated cells. Vascularization surrounding cells encapsulated inthin-film devices is important for function and survival of encapsulatedcells. To monitor the state of device vascularization, devices wereimplanted, then removed and imaged at 7, 14, 30 and 90 days (FIG. 6,Panels A-D). The first visible signs of vascularization of cellencapsulated thin-film devices were observed 14 days after implantation(FIG. 6, Panel B). These devices demonstrate a steady increase in vivovascularization of 1.5% daily over a 2-month period (FIG. 6, Panel E).Vascularization of these PCL devices occurred without any supplementaryadditional proangiogenic factors, as shown with implanted cell-freedevices with similar vascularization (FIG. 9, Panels A,B). When comparedwith common polymeric implant materials PLGA (FIG. 9, Panel C) and PVDF(FIG. 9, Panel D), PCL cell-free devices exhibited noticeably moredeveloped and branched vasculature. Furthermore, a relatively minimalforeign body response was observed (FIG. 10).

FIG. 11, Panel A depicts a PCL thin film device. The device includes twonanoporous layers in between which a lumen is defined. Each of thenanoporous layers have a microporous backing for structural support. Thetwo nanoporous layers are attached to each other at the periphery of thedevice all along the circumference, other than an inlet region at whichthe layers are not in contact with each other and define an opening intothe lumen of the device. A plastic tubing is inserted into the openingand held in place between the two layers. In addition, a third layer of1 mm-2 mm thick PCL layer is disposed along the periphery of the deviceto increase the rigidity at the periphery. FIG. 11, Panel B depictsanother embodiment of a PCL thin film device having two nanoporouslayers (each having a microporous backing) in between which a lumen isdefined.

FIG. 12 depicts the result of diffusion of IgG across a non-porouslayer, a 20 nm porous layer and a 200 nm porous layer. Three differentthin layer PCL devices were constructed. Each of the devices includestwo PCL layers attached to each other at the periphery of the device allalong the circumference, other than an inlet region at which the layersare not in contact with each other and define an opening into the lumenof the device. A non-porous membrane device was constructed using twoPCL layers into which nanopores or micropores were not introduced duringmanufacturing. A 20 nm porous membrane device was constructed using twonanoporous layers each having an average pore diameter of 20 nm andstructurally supported by a microporous backing layer. A 200 nm porousmembrane device was constructed using two microporous layers each havingan average pore size of 2 μm and a connectivity diameter of 200 nm. IgGwas loaded into each device and then the device was sealed shut, thedevice was then allowed to soak in a standard buffered saline solutionwhere the release of IgG from the device lumen to the external solutionthrough the porous layers was measured using a standard proteinmeasurement assay.

FIG. 13 illustrates that hES GFP-Insulin cells are viable for 40 days ineither a microporous or nanoporous thin film device prepared accordingto the methods disclosed herein. The microporous device used to generatethe data was made from two microporous PCL layers having ˜2 μm widepores formed in a ˜10 μm thick PCL film (the through channels in thefilm has a connectivity diameter of about 200 nm). The nanoporous deviceused to generate the data was made from two nanoporous PCL layers having30 nm-100 nm wide pores formed in a ˜10 μm thick PCL film. The devicewas subcutaneously implanted into the dorsal flank of BalbC mice.

FIG. 14 the same devices as used for FIG. 12 were utilized. Panel Adepicts c-peptide release from islet cells encapsulated in a 200 nmporous membrane device or a 20 nm porous membrane device. The isletcells in the device were exposed to a medium containing 2 mM glucose or20 mM glucose solution. The culture supernatant was collected at 30 minafter exposure to the 2 mM or 20 mM glucose. Insulin protein content inthe culture supernatant was measured using an enzyme linkedimmunosorbent assay (Mercodia). Panel B illustrates glucose stimulationindex (calculated as the ratio of insulin secreted at high-to-lowglucose conditions).

FIG. 15 shows foreign body response (FBR) to a thin film device madefrom polypropylene (Panel A; Grade 4) and from a PCL device madeaccording to the methods disclosed herein (Panel B; Grade 2). Thepolypropylene (PP) thin film device includes two thin layers of PP cutto the same size as the PCL device. The PP layers were non-porous. ThePCL device included two PCL layers, where one layer was microporous andthe other layer was nanoporous. The PP device was transplantedsubcutaneously into the dorsal flank of a BalbC mouse and assayed after2 weeks. The PCL device was transplanted subcutaneously into the dorsalflank of a BalbC mouse and assayed after 5 months.

FIG. 16 depicts a schematic of a thin film device having two openingsinto the lumen formed between the thin film layers. The two openingshold in place an inlet tube and an outlet tube. The inlet and outlettubes may be closed between use by using plugs, such as silicon plugs.The thin film device may also include a molecule (e.g., PD-L1 protein)to suppress the immune system.

The inventors have demonstrated the successful fabrication and use of aninnovative cell-encapsulating device that combines the some of thebenefits of both micro- and macro-encapsulation strategies. A flexiblethin-film geometry allows precise membrane porosity selection to directdesired cellular responses and interactions while maintaining a normalglucose response of encapsulated beta cells. A small reservoir volumeallows a rapid response to external stimuli, limiting dilutionalinterference from the device reservoir. Similar to micro-encapsulationdevices with large surface area to volume ratios, the thin-film devicestructure described herein is uninhibited by device thickness. Moreover,cells encapsulated in either micro- or nano-devices demonstrate aglucose stimulation index consistent with unencapsulated cells,indicating glucose sensing and responsive insulin secretion issuccessfully preserved. The devices described herein allow sufficientbioluminescence transmission through the device membrane to be measuredwith in vivo imaging systems. As demonstrated in vivo, the devicemembranes create a physical barrier between encapsulated cells and thehost environment, physically preventing cell contact initiatedsignaling. Furthermore, incorporation of a nano-porous membrane enablesthese devices to obstruct cytokine passage and protect encapsulatedcells from cytokine-mediated cell death. Additionally, in vivo studiesshow vascularization around the devices with limited fibrosis,suggesting great promise for this device as a long-term cellencapsulation device.

The thin film cell encapsulation devices for cell transplantationdescribed herein can be used to directly investigate the cellcontact-dependent or soluble factor-mediated signaling by controllingpore dimensions-inhibiting specific interactions. These devices have thecapacity to prevent immune cell contact with encapsulated cells, and thenano-porous device can protect encapsulated cells from cytokine-inducedcell death. Additional uses include using these devices in vivo toinvestigate modes of immune attack, whether contact- or solublefactor-mediated. These thin-film PCL devices have great potential asimplantable cell-encapsulation devices for treating diseases, such asType 1 Diabetes.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A device comprising an exterior surfacecomprising polycaprolactone (PCL) polymer, wherein said exterior surfaceis configured to be in contact with a bodily fluid or a bodily tissue ofa subject, and wherein, when said device is implanted in a subject forat least 5 months, a level of fibrosis around said device has a fibrosisscore of Grade 2 or less, as measured by histology, and wherein said PCLpolymer has a number average molecular weight (Mn) of 50-200 kDa.
 2. Thedevice of claim 1, wherein said exterior surface is coated with amolecule that promotes vascularization of said device, a molecule thatinhibits an immune response to said device, a molecule that inhibits aninflammatory response to said device, or any combination thereof.
 3. Thedevice of claim 1, wherein said exterior surface is coated with one ormore molecules selected from the group consisting of: vascularendothelial growth factor (VEGF), platelet-derived growth factor (PDGF),fibroblast growth factor (FGF-1), angiopoietin, monocyte chemoattractantprotein-1 (MCP-1), alpha_(v)beta₃, alpha_(v)beta₅, CD-31, VE-cadherin,ephrin, plasminogen activators, angiogenin, Del-1, acid fibroblastgrowth factor (aFGF), basic fibroblast growth factor (bFGF),follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocytegrowth factor (HGF), leptin, placental growth factor, platelet-derivedendothelial growth factor (PD-ECGF), and any combination thereof.
 4. Thedevice of claim 1, wherein said device further comprises a lumen.
 5. Thedevice of claim 4, wherein said lumen is configured to encapsulate aplurality of cells, a plurality of therapeutic molecules, or both. 6.The device of claim 5, wherein said device is configured such that saidplurality of therapeutic molecules are capable of diffusing out of saidlumen, out of said device, or both.
 7. The device of claim 5, whereinsaid plurality of cells are selected from the group consisting of: bonemarrow cells, mesenchymal stem cells, stromal cells, pluripotent stemcells, induced pluripotent stem cells, embryonic stem cells, bloodvessel cells, precursor cells derived from adipose tissue, bone marrowderived progenitor cells, intestinal cells, islets, Sertoli cells, betacells, progenitors of islet cells, progenitors of beta cells, peripheralblood progenitor cells, stem cells isolated from adult tissue, retinalprogenitor cells, cardiac progenitor cells, osteoprogenitor cells,neuronal progenitor cells, genetically transformed cells, and anycombination thereof.
 8. The device of claim 1, wherein said devicefurther comprises a first layer of biocompatible polymer and a secondlayer of biocompatible polymer.
 9. The device of claim 8, wherein saidfirst layer of biocompatible polymer, said second layer of biocompatiblepolymer, or both, comprise a plurality of nanopores.
 10. The device ofclaim 8, wherein said first layer of biocompatible polymer, said secondlayer of biocompatible polymer, or both, are selected from the groupconsisting of: methacrylate polymer, polyethyleneimine,polyethyleneimine-dextran sulfate, poly(vinylsiloxane)ecopolymerepolyethyleneimine, phosphorylcholine, poly(ethylmethacrylate), polyurethane, poly(ethylene glycol), poly(lactic-glycolicacid), hydroxyapatite, poly(lactic acid), polyhydroxyvalerte andcopolymers thereof, polyhydroxybutyrate and copolymers thereof,polycaprolactone, polydiaxanone, polyanhydride, polycyanocrylate,poly(amino acids), poly(orthoesters), polyesters, collagen, gelatin,cellulose polymer, chitosans, alginates, and any combination thereof.11. The device of claim 1, wherein said device is configured to beimplanted into said subject.
 12. The device of claim 11, wherein saiddevice is configured to be implanted into said subject by subcutaneousimplantation, omentum implantation, intraperitoneal implantation, orintramuscular implantation.
 13. The device of claim 11, wherein saiddevice is configured to be implanted into brain, spinal cord, pancreas,liver, uterus, skin, bladder, kidney, or muscle of said subject.
 14. Thedevice of claim 1, wherein said subject is selected from the groupconsisting of: a human, a monkey, a dog, a cat, a cow, a horse, a pig, asheep, a goat, a bear, a panda, a lion, a tiger, a leopard, an elephant,a zebra, a giraffe, a gorilla, a dolphin, and a whale.
 15. A methodcomprising implanting into a subject in need thereof a device accordingto claim 1, wherein said device further comprises a therapeuticallyeffective amount of one or more therapeutic molecules, thereby treatingsaid subject in need thereof.
 16. The method of claim 15, wherein saidsubject in need thereof has or is suspected of having diabetes mellitus.17. The method of claim 16, wherein said device further comprises atherapeutically effective amount of a plurality of insulin-secretingcells.
 18. The method of claim 15, wherein said device is implanted insaid subject in need thereof for at least 5 months.
 19. The method ofclaim 18, wherein said device does not elicit a significant foreign bodyresponse.
 20. The device of claim 1, wherein said device is circular,elliptical, square, rectangular, or a combination thereof.
 21. Thedevice of claim 1, wherein said device is fabricated from PCL polymer.